1. Field of the Invention
The present invention provides a sandwich immunoassay useful for detecting .beta.-N-acetylglucosaminidase (NAG) and a monoclonal antibody which recognizes NAG. The sandwich immunoassay comprises an immobilized anti-NAG monoclonal antibody and a labeled anti-NAG monoclonal antibody.
More particularly, the present invention provides a sandwich immunoassay for specifically detecting the presence of NAG isozyme B (and I) and a monoclonal antibody which recognizes NAG isozyme B (and I).
2. Discussion of the Background
.beta.-N-acetylglucosaminidase (NAG or .beta.-hexosaminidase, M.W.: approximately 120,000) is a widely distributed lysosomal enzyme, located predominantly in the renal proximal tubules. Increased NAG enzymatic activity in urine has been found to be associated with various kidney injuries. Currently, an assay for urinary NAG activity developed by Noto et al (Clin. Chem. (1983) 29, 1713) is routinely used for diagnosis and observation of rejection after renal transplantation, drug nephrotoxicity and acute renal insufficiency.
However, NAG exists as several isozymes, including A, B and I. In the urine of a normal healthy human, NAG isozyme A accounts for about 80-90% of the total isozymes while NAG isozyme B accounts for about 10-20%. NAG isozyme A is composed of two different subunits, .alpha. and .beta., whereas NAG isozymes B and I consist of two .beta.-subunits. These three isozymes have different isoelectric points, substrate specificities, thermal stabilities, and other physicochemical properties.
There have been many reports describing the clinical significance of NAG isozymes. For example, the ratio of NAG isozymes B and I rises in urine of patients with renal tubular injury caused by aminoglycoside antibiotics (Gibey et al, Clin. Chim. Acta (1984) 137, 1), pyelonephritis (upper urinary-tract infection) (Vigono et al, Clin. Chim. Acta (1983) 130, 297), renal transplantation (Yuen, Clin. Chim. Acta (1987) 164, 339), and other similar disorders. The elevation of NAG isozyme I has been reported in blood of patients with hepatitis, leukemia, and other such diseases. A specific assay for NAG isozymes B and I would have increased sensitivity for the diagnosis of renal damage compared with existing assays which detect the total amount of all NAG isozymes.
The separation of NAG isozymes has been performed utilizing a variety of techniques including: ion-exchange chromatography (Ellis et al, Clin. Chim. Acta (1975) 64, 195), electrophoresis (Pitkanen et al, Enzyme (1982) 28, 14), and heat treatment (Oberketter et al, Clin. Chim. Acta (1979) 94, 281). However, these methods are difficult to adopt to routine clinical work because they are complicated, imprecise, and poorly sensitive toward NAG separation.
Recently, an enzyme immunoassay (EIA) using monoclonal antibodies specific for NAG isozyme A and B was reported (Isaksson et al, Scand. J. Clin. Lab. Invest. (1989) 49, 597-603). In this antigen capture assay, NAG is immunologically bound to an immobilized anti-NAG monoclonal antibody, and the enzymatic activity of bound NAG is measured. Unfortunately, this assay is not quantitative because the anti-NAG monoclonal antibodies inhibit the enzymatic activity of NAG.